We are using two approaches to identify and isolate genes involved in the regulated growth and differentiation of the mammalian embryo and fetus. The first of these is insertional mutagenesis, in which mutant phenotypes are generated by the insertion of exogenous DNA into a gene. This also creates a molecular tag at the mutant locus, providing direct access to the gene. For several years, we have analyzed transgenic mouse strains derived from embryonic stem (ES) cells deliberately infected in tissue culture with retroviruses to generate insertional mutations. We screened over 40 proviral insertions specifically for mutations that lead to prenatal lethality, indicating that the gene is absolutely essential for normal development. This screen identified 4 recessive prenatal lethal mutations tightly linked to, and presumably caused by, proviral insertion. Currently we are focusing on the insertional mutation caused by the 412-r provirus. We isolated the 412-r retroviral insertion site and surrounding DNA in cosmids. Fragments of candidate genes were obtained using exon-trapping, extended by 5' and 3' RACE, and used for probing embryo cDNA libraries. These efforts allowed us to identify a candidate for the mutated gene. The provirus lies in the first intron of the gene for Sumo/sentrin specific protease 1 (SENP1), a recently identified protein involved in the regulation of Sumoylation. Sumo/sentrin is a small protein related to ubiquitin that is added to proteins in a process similar to ubiquitination. SENP1 acts to remove Sumo from modified proteins. Our evidence for 412-r mutating the SENP1 gene comes from the analysis of expression levels in homozygotes, which have an approximately 50% reduction. SENP1 is normally ubiquitously expressed. Either the viral insertion causes a reduced expression level in all cells or completely down-regulates the gene in only some cell or tissue types. Currently we are assessing this by in situ hybridization of mutant embryos. Our ongoing analysis of the 412-r mutant phenotype will provide insight into where and when SENP1 function is critically important for the embryo. Preliminary results suggest roles in craniofacial development. Complete elucidation will provide important new insights into the biological functions of SENP1 and Sumoylation during development. We also have developed an approach to identify developmentally important molecules that might elude screening strategies based on differential mRNA expression. Rapid protein degradation by the ubiquitin/proteasome pathway is of critical importance in many key cellular processes, including cell cycle progression and programmed cell death. We hypothesized that the activity of many critical regulatory molecules in development is regulated by ubiquitin-mediated protein degradation, and that these molecules can be identified by their interaction with E3 ubiquitin ligases, the component of the ubiquitination pathway conferring specificity. We used the developmentally regulated E3 Nedd-4 in a yeast two-hybrid screen of genes expressed at midgestation and isolated 4 candidates, Nedd-4 Binding Proteins (NBP) 1-4. Three of these are novel genes and the fourth encodes PLIC2, a protein that mediates interactions between CD47 and the cytoskeleton. Further experiments showed that NBP1 and -2 are ubiquitinated in vitro by Nedd-4. We have preliminary evidence that NBP2 is ubiquitinated in vivo as well. Ultimately, we are interested in the key question of whether NBP1-4 are crucial for development and specifically what role their interaction with the ubiquitination process, including the potential for rapid degradation, might play. We are carrying out in vitro mutagenesis to identify regions that confer binding of NBP1-4 to Nedd-4. Mutations that prevent binding to Nedd-4 may uncouple these proteins from the ubiquitination process.